Head and neck cancer is the sixth most common cancer worldwide. Tobacco and/or alcohol are involved in approximately 75% of all human squamous cell carcinomas of the head and neck (SCCHN). The overall survival rate for human SCCHN (approximately 50% in five years) has not changed very much in recent decades, so there is an urgent need for new approaches for SCCHN treatment. We have developed a novel carcinogen induced murine oral cavity and esophageal carcinogenesis model. We will use this 4-nitroquinoline oxide (4-NQO) carcinogenesis model to measure the effects of alcohol (ethanol) on the incidence of oral cavity carcinogenesis and on epigenetic changes. We will test our hypothesis that alcohol may contribute to carcinogenesis in epithelial stem/progenitor cells of the basal layer of the tongue by promoting epigenetic changes, such as histone modifications or greater DNA methylation at CpG cytosines, which leads to gene silencing. A corollary of our hypothesis is that ethanol leads to aberrant epigenetic changes because ethanol lowers the levels of retinoic acid (RA) in cells via inhibition of RA production from retinol, and we've shown that RA is a signaling molecule which has a major role in initiating epigenetic changes during stem cell differentiation. The specific aims of the application are: (1) to measure the effects of alcohol on the incidence of oral cavity cancer in our murine oral cavity carcinogenesis model, and to assess the expression of molecular markers such as cyclin D1; RAR22; p16; SFRP 1,2,4, and 5; and nanog in normal epithelial stem/progenitor cells versus cancer stem/progenitor cells from the tumors by immunohistochemistry; (2) to assess epigenetic changes in epithelial cells in the basal layer of oral cavity tissues, such as the tongue, during 4-NQO carcinogenesis, with and without subsequent alcohol administration. We will purify both normal cells from the basal layer of the tongue and cells with properties of cancer stem/progenitor cells by FACS and measure epigenetic markers by the chromatin immunoprecipitation (ChIP) and the ChIP-Chip techniques; and (3) to determine if drugs that inhibit specific epigenetic modifications influence the incidence of oral cavity cancer and/ or influence the epigenetics of basal layer stem/progenitor cells during the carcinogenesis process in the presence of alcohol, again primarily using ChIP and ChIP-Chip approaches. These proposed experiments will provide important insights into the molecular mechanisms by which alcohol influences carcinogenesis. They will also test the importance of epigenetic changes in stem/progenitor cells in the development of SCCHNs and explore the mechanisms by which drugs that modify epigenetic changes act therapeutically. The proposed experiments will help to establish the roles of stem/progenitor cells in the epithelium during the oral cavity carcinogenesis process. We will gain valuable information about how the epigenome of stem/progenitor cells is influenced by carcinogens, alcohol, and drugs, such as zebularine and 5-aza-2'-deoxycytidine, which have been shown both to inhibit DNA methyltransferases and to reduce tumor incidence in other carcinogenesis models. )